This proposal represents a logical extension of ongoing research on the structure and function of the chromosome of Mycobacterium tuberculosis. The overall objective of these studies is to analyze the sites of IS6110 insertion in the M. tuberculosis chromosome. A comparison of sites of insertion will distinguish between those that are common and those that are unique. In addition to providing basic information about these sites, unique insertion site sequences will be used as targets in a polymerase chain reaction (PCR) assay which will identify specific strains of M. tuberculosis associated with ongoing outbreaks, for example, drug- resistant outbreaks. This rapid method will accelerate the initiation of proper antituberculosis therapy. Common sites of insertion will be used to subgroup strains and perhaps identify specific geographic origins. Subgrouping will provide a means for studying transmission of tuberculosis among patients in a defined geographic area. The specific aims are: 1) To determine if there are highly conserved sites of insertion of IS6110 in the genome of M. tuberculosis in addition to the single site of insertion which is highly conserved in the genome of M. tuberculosis and M. bovis. This will be accomplished by developing an indirect hybridization technique which localizes insertion sites in cloned fragments and using sequences from these fragments in PCR assays for testing large numbers of strains. 2) To determine if unique sites of insertion of IS6110 can be used for rapid identification of specific strains of M. tuberculosis, e.g., specific multidrug-resistant strains. This will involve identifying insertion sites that are specific for a multidrug-resistant strain associated with an ongoing outbreak and developing a PCR assay to detect those insertion sites. 3) To determine if isolates from a defined geographic area share common site(s) of insertion of IS6110. This will involve identifying insertion sites in isolates from the Texas/Mexico border and comparing them to insertion sites in isolates from other widely-distributed areas of the U.S. 4) To construct a large restriction fragment map of the M. tuberculosis chromosome that can be used to locate the sites of insertion of IS6110. This will be accomplished by pulse-field gel electrophoresis and the construction of specific linker clones.